US5882933A - Method for determination of leukocytes and hemoglobin concentration in blood - Google Patents

Method for determination of leukocytes and hemoglobin concentration in blood Download PDF

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US5882933A
US5882933A US08/898,000 US89800097A US5882933A US 5882933 A US5882933 A US 5882933A US 89800097 A US89800097 A US 89800097A US 5882933 A US5882933 A US 5882933A
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subpopulations
cell sample
blood cell
sample
blood
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Yi Li
Carole Young
Robert H. Raynor
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Beckman Coulter Inc
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Priority to EP98932979A priority patent/EP1000355A1/de
Priority to PCT/US1998/013553 priority patent/WO1999005523A1/en
Priority to JP2000504458A priority patent/JP2001511525A/ja
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G01N33/721—Haemoglobin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • G01N15/12—Investigating individual particles by measuring electrical or magnetic effects by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5094—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1402—Data analysis by thresholding or gating operations performed on the acquired signals or stored data
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1477—Multiparameters
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/824—Immunological separation techniques
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/101666—Particle count or volume standard or control [e.g., platelet count standards, etc.]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/107497—Preparation composition [e.g., lysing or precipitation, etc.]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25125—Digestion or removing interfering materials
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/2525—Stabilizing or preserving

Definitions

  • the present invention relates to a method to enable a determination of leukocyte subpopulations in a blood cell sample by means of suitable electronic instrumentation.
  • the present invention relates to a determination of hemoglobin concentration in said blood sample.
  • Analysis of leukocyte subpopulations from whole blood samples is an integral and essential part of diagnostic procedures regarding a multiplicity of pathologies.
  • the ability to analyze the major subpopulations of leukocytes in an automated manner is essential for a rapid diagnosis of a single blood sample and for the rapid processing of many samples at once.
  • the determination of hemoglobin concentration provides useful diagnostic information about the health of a patient.
  • U.S. Pat. No. 4,286,963 (to Ledis et al.) describes a method for achieving rapid hemolysis of red blood cells in whole blood and automated analysis of lymphoid and myeloid subpopulations of leukocytes, and quantitative determination of hemoglobin using potassium cyanide.
  • U.S. Pat. No. 4,485,175 (to Ledis et al.) describes a method for performing differential determinations of leukocytes into three (3) subpopulations utilizing automated cell counting equipment. However, this method is limited to effect differentiation of the leukocytes into three subpopulations: lymphocytes, monocytes and granulocytes.
  • U.S. Pat. No. 5,155,044 discloses a method for isolation and analysis of leukocytes from a whole blood sample, which enables automated differentiation of leukocytes into five (5) subpopulations utilizing an automated hematology analyzer.
  • this method cannot convert oxyhemoglobin of the sample to a stable chromogen for hemoglobin measurement.
  • the oxyhemoglobin method is an unreliable method because of incomplete hemoglobin conversion.
  • U.S. Pat. No. 5,389,549 (to Hamaguchi et al.) describes a lysis reagent system which contains a nonionic polyoxyethylene surfactant.
  • a lysis reagent system which contains a nonionic polyoxyethylene surfactant.
  • Full analysis of leukocyte subpopulations requires differential lysis of the red blood cells and leukocytes, and three separate determinations for the identity of eosinophil, neutrophil and basophil populations in addition to the lymphocyte and monocyte populations. Additionally, this system requires a hypotonic lysing environment which is extremely shocking to the cells and makes preservation of the cells in a near native state difficult.
  • measuring hemoglobin concentration in a blood sample is another diagnostic tool when doing blood analysis.
  • hemoglobin determinations have been performed by forming and measuring cyanide hemoglobin.
  • the reagent waste from this method is of enormous environmental concern.
  • cyanide-free methods for lysing red blood cells and measuring hemoglobin have been developed.
  • U.S. Pat. No. 5,250,437 (to Toda et al.) and U.S. Pat. No. 5,242,832 (to Sakata) all utilize quaternary ammonium salt lysis systems for hemolyzing red blood cells and oxidizing the hemoglobin.
  • EPO No. 0 325 710 discloses a method for the hemolysis of red blood cells. However, with this method, one can only differentiate three subpopulations in addition to measuring the oxyhemoglobin.
  • U.S. Pat. No. 5,516,695 discloses a method for rapid analysis of a whole blood sample allowing the determination of five subpopulations of white blood cells, nucleated red blood cells, and lymphocyte immunophenotyping on automated hematology instrumentation.
  • the disclosed method lyses red blood cells and concurrently fixes white blood cells and preserves surface antigens on lymphocytes.
  • the preservation of white cell surface marker is based on fixation by an aliphatic aldehyde, which is known as an environmentally unfriendly chemical.
  • the method requires heating of the reagent to about 40° C. for leukocyte differential of a whole blood sample in order to achieve the performance and the throughput requirement of an automated hematology analyzer. Without heating, the lysing time is substantially longer.
  • U.S. Pat. Nos. 5,030,554 and 5,437,985 disclose a method to prepare a whole blood sample for photo-optical analysis of lymphocyte subpopulations.
  • the method comprises a 3-step sample preparation of: (1) lysing red cells with an acid lyse; (2) quenching the lysing reagent by an alkaline quench solution; (3) fixing white cells with a fixative solution. Indicator binding can be accomplished before, concurrent with, and after the lysing.
  • This method also utilizes aldehyde fixation to preserve white cells.
  • the light scatter and fluorescence analyses as disclosed differentiate only three leukocyte subpopulations, i.e., monocytes, lymphocytes and granulocytes.
  • one object of the present invention is to provide a method for counting WBC, determining hemoglobin concentration, and differentiating at least four subpopulations of leukocytes in a blood cell sample.
  • the method comprises exposing a blood cell sample to a lytic reagent for a time sufficient to lyse red blood cells without heating said exposed blood cell sample; adding a non-aldehyde stabilizing reagent to said exposed blood sample, wherein said stabilizing reagent inhibits further lytic action and stabilizes leukocytes without fixing the cells; analyzing said stabilized sample in less than 30 seconds after addition of the lytic reagent, wherein the mode of analysis is selected from at least two modes selected from DC, RF, and light scatter, wherein said analyzing is for counting WBC, and differentiating at least four leukocyte subpopulations selected from lymphocytes, monocytes, basophils, neutrophils and eosinophils; measuring light absorbance of the said stabilized sample at about 540 nm;
  • Another object of the present invention is to provide a method for stromatolysis of red blood cells in a blood cell sample, analysis of leukocyte subpopulations and lymphocyte subpopulations, and determining hemoglobin concentration in the blood cell sample.
  • the method comprises incubating a blood sample with a fluorochrome-conjugated antibody; exposing of the incubated blood sample to a lytic reagent for a time sufficient to lyse red blood cells without heating said exposed blood cell sample; adding a non-aldehyde stabilizing reagent to said exposed blood sample, wherein said stabilizing reagent inhibits further lytic action and stabilizes leukocytes of the said blood sample in hypertonic medium ranging from about 400 mOsm to about 600 mOsm without fixing the cells; analyzing said stabilized sample in less than 30 seconds after addition of the lytic reagent, wherein the mode of analysis is selected from at least two modes selected from DC, RF, light scatter and fluorescence, wherein said analyzing is for differentiating
  • a further object of the present invention is to provide a method for stromatolysis of red blood cells in a blood cell sample, analysis of leukocyte subpopulations and lymphocyte subpopulations, and determining hemoglobin concentration in the blood cell sample.
  • the method comprises incubating a blood sample with antibody-conjugated latex particles; exposing the incubated blood sample to a lytic reagent for a time sufficient to lyse red blood cells; adding a non-aldehyde stabilizing reagent to said exposed blood sample, wherein said stabilizing reagent inhibits further lytic action and stabilizes leukocytes of the said blood sample without fixing the cells; analyzing the said stabilized blood sample in less than 30 seconds after addition of the lytic reagent, wherein the mode of analysis is selected from at least two modes selected from DC, RF, and light scatter, wherein said analyzing is for differentiating leukocyte subpopulations; measuring light absorbance of said stabilized sample at about 540 nm; reporting leukocyte subpopulations in
  • the invention is particularly advantageous vis-a-vis the prior art discussed above in that the invention provides a method which preserves cell surface morphology and enables lymphocyte immunophenotyping in addition to differentiation of leukocyte subpopulations.
  • Another advantage of the present method is that it provides an automated hemoglobin concentration determination in conjunction with the above referenced differentiation and immunophenotyping.
  • FIGS. 1-10 and 15-16 are scattergrams obtained in accordance with the practice of the present invention as described in Examples III, IV, V and VI;
  • FIG. 11 is a graph illustrating the absorption profile of the sample described in Example VII.
  • FIGS. 12-14 are scattergrams obtained in accordance with the practice of the present invention as described in Examples III and IV;
  • FIG. 17 is a curve illustrating the correlation between results obtained by a reference method and the method of the present invention as described in Example X;
  • FIG. 18 is a scattergram obtained in accordance with the practice of the present invention as described in Example XI.
  • a blood sample can be obtained from a patient by conventional phlebotomy techniques.
  • anti-coagulant agents including heparin, EDTA, acid citrate dextrose (ACD) and sodium citrate do not affect the performance of the present invention.
  • the blood sample is briefly mixed with a lytic reagent composition.
  • the lytic reagent composition effectively stromatolyses red blood cells without detrimentally affecting leukocytes for subsequent analysis.
  • the amount of time of exposing the blood sample to the lytic reagent composition prior to addition of a stabilizing reagent composition is important for the differentiation of leukocyte subpopulations presented by this invention. This exposure period should not exceed ten seconds, and is preferably less than seven seconds.
  • the exposure time is specified for ambient temperatures (18° to 28° C.).
  • This fast lytic activity preserves the leukocytes in near native conditions by avoiding prolonged exposure to the lytic reagent.
  • near native condition means that cellular morphology is preserved so that analysis of the cellular subpopulations can be performed using histochemical or fluorescent labelling of cell surface markers.
  • a unique feature of the present method is that red blood cell lysis can be performed at ambient temperatures and it does not require heating of the mixed sample.
  • a non-aldehyde stabilizing reagent composition which functions to inhibit lytic activity and prevent swelling of leukocytes so that the leukocytes are preserved for automated analysis, including differentiation of leukocytes and immunophenotyping of the lymphocytes.
  • the leukocyte fraction of the whole blood sample, treated by the above procedure, can be readily differentiated into at least two subpopulations of leukocytes.
  • at least three subpopulations and more preferably at least four subpopulations and most preferably at least five subpopulations are differentiated, which include neutrophils, lymphocytes, monocytes, eosinophils and basophils.
  • a lytic reagent composition and stabilizing reagent composition form a lytic reagent system.
  • a single step measurement means that a single aliquot of blood can be used with the same lytic reagent system to obtain a differentiation of a lymphocyte subpopulation with at least one other subpopulation of leukocytes consisting of eosinophils, basophils and neutrophils. Preferably, this differentiation is measured in less than 30 seconds and more preferably less than 20 seconds after the addition of the lytic reagent composition.
  • Previous lysis reagent systems permitted only two parameter analyses in a given analysis step. Thus, to fully obtain a profile of five leukocyte subpopulations, a complex method of three individual determinations on the same sample followed by a combined analysis of the determinations was required.
  • the method of the present invention provides the advantage of operating entirely at room temperatures, 18° to 28° C. Previous methods operated at an elevated temperature, 30° C. or higher, for adequate separation of eosinophils and basophils. This elevated temperature requirement necessitated analysis instrumentation which was significantly more complex, as the reactions must be thermostatically controlled.
  • the present invention overcomes this need for thermostatic control by operating optimally at room temperature.
  • hemoglobin concentration in blood samples is an essential part of diagnostic analysis and is also important for monitoring responsiveness to therapies directed towards diseases which affect hemoglobin and to therapies which are directed towards other diseases but which may have adverse side effects on the hemoglobin level.
  • the present invention allows for the analysis of at least three, preferably four, and more preferably five subpopulations of leukocytes in conjunction with a determination of hemoglobin concentration.
  • Lysis of red blood cells with the lytic reagent composition causes the release of hemoglobin as oxyhemoglobin.
  • Addition of the stabilizing reagent composition results in the formation of a stable chromogen which has a maximum absorbance peak at 540 nm and a shoulder at 570 nm.
  • the present method provides several advantages over the methods of hemoglobin measurement of the prior art. Unlike the previous methods, the present invention allows for the differentiation and analysis of leukocyte subpopulations in their near native state along with a determination of the hemoglobin concentration. In addition, the method provides for converting oxyhemoglobin to a stable hemoglobin chromogen in less than 10 seconds which allows for rapid automated analysis. The chromogen once formed is stable for up to 20 minutes.
  • cell surface markers can be labeled by a indicator, such as fluorochrome-conjugated antibodies, fluorescence dyes, and antibody-conjugated particles by incubation or staining.
  • a indicator such as fluorochrome-conjugated antibodies, fluorescence dyes, and antibody-conjugated particles by incubation or staining.
  • labeling of cell surface antigens with various fluorochrome-conjugated antibodies allows for lymphocyte immunophenotyping on conventional flow cytometers using the method of the present invention.
  • Example X shows an example of differentiation of CD8 positive lymphocytes using the method of the present invention on a fluorescence flow cytometer.
  • commercial fluorescence flow cytometers require a separate sample preparation prior to transferring the sample to a flow cytometer for analysis.
  • the method of the present invention can be used in a flow cytometer in an automatic mode because of the rapid speed and simplicity of the process.
  • fluorescence flow cytometers utilize photooptical measurements, which typically measure forward and side light scatters and fluorescence signals.
  • the flow cytometers are routinely used for lymphocyte immunophenotyping.
  • their capability in leukocyte differential is usually limited to a 3-part differential, i.e., monocytes, lymphocytes and granulocytes.
  • cell concentrations after such pre-treatments no longer correlate to their original concentrations in the blood. Therefore, only percentages of leukocyte subpopulations are reported from the instrument, and not an absolute count of an interested cell type of the blood sample. Consequently, red blood cell count, white blood cell count, and leukocyte differentials are measured in hematology analyzers or blood counters.
  • hematology methods use a lytic reagent for lysing red blood cells that is harsh to the leukocytes, because the lytic reagent either rips off the cell membrane or damages the cell membrane to a degree necessary to achieve differentiation of different cell types.
  • the methods which use these lytic reagents are not compatible with immunophenotyping because of their inability to preserve cell surface markers. Therefore, hematology and immunology analyses of a blood sample are routinely performed on two different instruments, i.e., a hematology analyzer and a fluorescence flow cytometer, and most likely in two different clinical laboratories.
  • the present method provides a unique opportunity to combine leukocyte differential analysis with lymphocyte immunophenotyping, especially for the most commonly desired T and B cell types.
  • This method can be performed in a single instrument in an automated mode. Since no additional cell separation and pre-treatments are needed using the method of the present invention, one can obtain both an absolute count of total white blood cells (WBC) and absolute count of leukocyte subpopulations when a known volume of a blood sample and known volumes of reagents are delivered.
  • WBC total white blood cells
  • Another advantage of the present method is preservation of leukocyte morphology and cell surface markers without using a fixative.
  • blood sample preparations for immunophenotyping involve multiple steps of cell separation, washing, fixing and resuspending in an appropriate medium. It is known from the literature that cell damage or loss of a particular cell type is common after such lengthy sample preparation. Moreover, as previously discussed, obtaining absolute count for WBC and leukocyte subpopulations becomes impossible unless other secondary means are employed, such as introducing reference particles as taught by Shenkin, et al. in U.S. Pat. No. 5,451,525.
  • fixatives such as aldehydes are toxic. Although aldehydes are effective in preserving cell surface markers, the waste generated from hematology analyzers using aldehyde containing reagents cause environmental concerns.
  • the method of the present invention selectively lyses red blood cells and preserves leukocyte cell morphology and surface markers without fixing the cells. Compared to conventional sample preparation methods and reagents, the method of the present invention is rapid, simple, and especially suitable for use on automated hematology analyzers and flow cytometers in an automated mode.
  • Example XI shows an alternative mode of lymphocyte subpopulation analysis by using non-fluorescence electro-optical analysis.
  • CD4 positive lymphocytes (CD4+) are labeled with antibody-conjugated latex particles.
  • the labeled blood sample is treated by the method of the present invention as described in Example XI, the CD4+ cells are differentiated by their shifted light scatter signals.
  • the analysis is performed on a hematology analyzer equipped with DC, RF and light scatter measurement devices for differentiating leukocytes using the same lytic reagent system.
  • the CD4 analysis is performed by switching from a 5-part differential analysis mode to a CD4 and CD8 lymphocyte analysis mode. The same reagent volumes and reaction times are used for both analysis modes.
  • This example further illustrates the ability of the method of the present invention for further analysis of lymphocyte subpopulations.
  • One lytic reagent composition that is suitable in the above described method comprises an ethoxylated long chain amine compound and an acid to adjust the pH of the composition.
  • the ethoxylated long chain amine compound has a lipophilic tail and a branched hydrophilic, polar head group and can be represented by the formula: ##STR1## wherein R is an alky, alkenyl or alkynyl having 12 to 22 carbon atoms; m and n are each 1 or more, and m+n is 20 to 40. In the above structure, R is preferably 14 to 20 carbon atoms and m and n can be the same or different numbers.
  • the ethoxylated long chain amine compound of formula (I) can be synthesized by procedures known in the art as described, for example, in Porter, M. R., Handbook of Surfactants, Chapman & Hall, NY, pp. 147-150 (1991).
  • m and n in the above formula should have approximately equal values. However, m and n need not have the same values.
  • the concentration of the ethoxylated long chain amine compound in the lytic reagent composition needs to be in an amount sufficient to selectively hemolyze the red blood cells in a whole blood sample, while leaving the remaining leukocytes essentially intact.
  • the concentration of the ethoxylated long chain amine compound in the lytic reagent composition has been found to be effective in a broad range from about 8 g/L to about 80 g/L, preferably 12 g/L to 50 g/L.
  • the acid is used in an amount sufficient to adjust the pH of the lytic reagent composition in the range of approximately 2.0 to 3.6.
  • the acid will usually be an effective amount of an organic acid.
  • formic acid or an effective mixture of formic acid with another organic acid or an inorganic acid is used.
  • the organic acid to be used in admixture with the formic acid can be, for example, acetic, citric, oxalic, glycolic or propionic acid or a mixture of two or more of the aforementioned acids.
  • Inorganic acids which can be mixed with the formic acid include, but are not limited to, hydrochloric, sulfuric and phosphoric acid.
  • the stabilizing reagent composition is added subsequent to red blood cell lysis to inhibit further lytic activity.
  • Another unique feature of the present method is the use of a non-aldehyde stabilizing reagent composition.
  • the non-aldehyde stabilizing reagent composition functions to neutralize the acid in the blood mixture and prevent swelling of leukocytes so that the leukocytes are preserved for the purposes of automated analysis, including differentiation of leukocytes and immunophenotyping of lymphocytes.
  • the stabilizing reagent composition is an aqueous buffered salt solution comprised of a simple physiological salt or salts.
  • the salt or salts used in the stabilizing reagent composition can be a mixture of chloride salts and sulfate salts.
  • the chloride salt can be, but is not limited to, sodium chloride or potassium chloride in a concentration of about 0.25 to 4% based on the total weight of the stabilizing reagent composition.
  • the sulfate salt can be, but is not limited to, sodium sulfate or potassium sulfate in a concentration of about 0.25 to 9% based on the total weight of the stabilizing reagent composition.
  • the stabilizing reagent composition is hypertonic and can have an osmolality of about 950 to 1800 mOsm.
  • the salt concentration which affects the osmolality of the stabilizing reagent composition can vary because the volume of the stabilizing reagent composition can be adjusted relative to the lytic reagent volume so that the final osmolality of the blood sample mixture is between approximately 400 to 600 mOsm, preferably 410 to 520 mOsm.
  • the buffer may be any physiological buffer including, but not limited to, potassium or sodium carbonate, potassium or sodium phosphate, Tris, and potassium or sodium tetraborate.
  • the pH of the stabilizing reagent composition is an approximate pH of 7 to 12.5, preferably having a pH of 9 to 11.5.
  • one or more additive can be included in the lytic reagent system in concentrations that their presence is compatible with the primary functional components of the lytic reagent system.
  • these additives are solubilizers and preservatives.
  • a slight hypertonic condition is preferred, instead of a physiologically isotonic environment.
  • the hypertonic environment created by the stabilizing reagent composition by its high physiological salt content prevents the swelling of the leukocytes that would result from their exposure to the lytic reagent composition and prevents the cell damages due to such swelling.
  • a slight cell shrinkage occurs upon interaction with the stabilizing reagent for a few seconds, which produces a more confined cell distribution among the leukocyte subpopulations.
  • a hypotonic environment is not required by the current method for the lysing of red blood cells.
  • the addition of the stabilizing reagent composition converts the test sample to a slightly hypertonic environment which does not produce a dramatic osmotic stress to the leukocytes.
  • the hypertonic environment ranges from 400 to 600 mOsm. The method of this invention results in a much tighter distribution within each subpopulation and a superior population separation when compared to the performances of prior art methods.
  • the leukocytes of aged and abnormal blood samples are usually fragile or sensitive to lysing conditions and are difficult to analyze by automated blood analyzers.
  • the harshness of most lysis reagent systems, particularly acid based lysis systems precludes their use for analysis of any but fresh blood samples, as the cells become too fragile as they age.
  • the advantage of the present method for preventing leukocyte damage not only allows for differential analysis of freshly collected blood samples, but also for analysis of blood samples several hours after sample collection and also abnormal blood samples.
  • Another advantageous feature of this invention is its utility for differential analysis of other fluid samples, such as bone marrow, and blood samples of non-human species.
  • a first lytic reagent composition has been formulated with the following composition:
  • a second lytic reagent composition has been formulated with the following composition:
  • BASF Pluronic 25R8
  • a third lytic reagent composition has been formulated with the following composition:
  • BASF Pluronic F38
  • a first stabilizing reagent composition has been formulated which is comprised of 14 g/L of NaCl, 32 g/L of Na 2 SO4, and 6.6 g/L of Na 2 CO 3 buffer, pH adjusted to 11.0.
  • the osmolality of this reagent is about 1080 mOsm.
  • a second stabilizing reagent has been formulated and contains 8.3 g/L Na 2 HPO 4 , 12.2 g/L Na 3 PO 4 , 14.3 g/L NaCl and 31 g/L Na 2 SO 4 , pH adjusted to 11.
  • the osmolality of this reagent is about 1190 mOsm.
  • a lytic reagent system prepared in deionized water from reagent grade chemicals and ethoxylated long chain amine compounds of industrial purity.
  • Example I a 20 g of the cationic ethoxylated long chain amine compound described in Example I a) was dissolved in 1 L of water. The pH of the ethoxylated long chain amine compound solution was adjusted to 3.2 by formic acid. 0.2 g of EDTA and 0.5 g of Proclin 300 (Rohm and Haas Co.) were added as antioxidant and anti-microbial preservatives, respectively. To 31 ⁇ l of a whole blood sample, 560 ⁇ l of the lytic reagent composition was added and the mixture was gently mixed by swirling for 4 seconds at room temperature (approximately 21° C.).
  • the lysing reaction was retarded by addition 230 ⁇ l of an aqueous stabilizing reagent composition containing 14 g/L of NaCl, 32 g/L of Na 2 SO 4 and 6.6 g/L of Na 2 CO 3 , pH 11.0.
  • the blood mixture was gently mixed and ready for differential analysis 15 seconds after the addition of the stabilizing reagent.
  • the final blood mixture was kept at neutral pH (about 7) and in hypertonic condition with a osmolality about 445 mOsm.
  • Three-dimensional differential analysis was conducted on a COULTER® STKS hematology analyzer with DC, RF and light scatter measurements utilizing a focus flow technique and ISOTON® III diluent as a sheath fluid.
  • FIG. 1 and FIG. 2 The resulting scattergrams are illustrated in FIG. 1 and FIG. 2.
  • FIG. 1 Four distinct subpopulations of leukocytes were identified and quantified in the DC vs. rotated light scatter (RLS) scattergram, FIG. 1.
  • FIG. 2 illustrates the separated leukocyte subpopulations in the scattergram of DC vs. Opacity (a function of RF and DC).
  • a fifth subpopulation of the leukocyte, basophils is isolated by gating out other overlapping subpopulations in the DC vs. Opacity scattergram.
  • the isolated basophil population is depicted in the scattergram illustrated in FIG. 3.
  • FIG. 14 shows four of the leukocyte subpopulations, including lymphocytes, monocytes, neutrophils and eosinophils.
  • FIG. 15 shows four leukocyte subpopulations seen with the DC vs. light scatter scattergram obtained from this analysis.
  • a fifth leukocyte subpopulation, basophils, can be obtained by gating the acquired data as described above.
  • FIG. 16 shows the DC vs. light scatter scattergram obtained from this analysis. This scattergram distinctly shows four of the leukocyte subpopulations including lymphocytes, monocytes, neutrophils and eosinophils.
  • the basophils can be obtained by gating the acquired data as described above.
  • Example III The procedure of Example III was repeated for leukocyte differentiation of blood from an active sickle cell patient, using the same reagents as in Example III. It has been commonly seen that active sickle cell blood is difficult to lyse by some commercial hypotonic acid lysing reagents and the subsequent population separation among the leukocyte subpopulations is poor. In these cases, a false differential report can be generated by an automated hematology analyzer. As shown in FIG. 4, a clear population separation indicating the presence of pathology by the abnormality leukocyte differentials was obtained for the active sickle cell blood sample by using the method of this invention.
  • Example III The procedure of Example III was repeated for leukocyte differentiation of blood from a patient having acute lymphocytic leukemia. As seen in FIG. 12, analysis of the blood sample using the method of this invention indicates the presence of atypical lymphocytes.
  • FIGS. 5 to 9 show Several veterinary whole blood samples with following order: FIG. 5, canine whole blood sample; FIG. 6, simian whole blood sample; FIG. 7, caprine whole blood sample; FIG. 8, equine whole blood sample and FIG. 9, guinea pig whole blood sample.
  • the leukocyte subpopulations including lymphocytes, monocytes, neutrophils and eosinophils within a species, are clearly distinct from each other.
  • the lytic reaction time and reagent volume can be varied in order to obtain the best differential results, but such variations can be easily accomplished by automated blood analyzers.
  • This invention allows, for the first time, an ability to differentiate at least four different subpopulations of leukocytes, i.e., lymphocytes, monocytes, neutrophils and eosinophils, with veterinary whole blood samples utilizing an automated method.
  • leukocytes i.e., lymphocytes, monocytes, neutrophils and eosinophils
  • Example III The procedure of Example III was repeated utilizing the same lytic reagent system for leukocyte differentials of a whole blood sample several hours after gathering for direct comparison of the differentiation with fresh blood samples.
  • the sample was stored at room temperature, approximately 21° C.
  • FIG. 10A Similar leukocyte subpopulation profiles were obtained for fresh blood, FIG. 10A, and blood samples that were 27 hours old, FIG. 10B, demonstrating that this invention can be used for leukocyte differentiation and analysis several hours after blood sample collection.
  • the lytic reagent system of Example III was used in the determination of hemoglobin concentration in whole blood samples. 14 ⁇ l of whole blood were mixed with 1224 ⁇ l of the lytic reagent composition and gently mixed for 5 to 8 seconds. 498 ⁇ l of the stabilizing reagent composition was added and after 10 seconds, an absorption profile of the resulting chromogen was measured. As seen in FIG. 11, the chromogen has a maximum absorption peak at 540 nm with a shoulder at 570 nm. The chromogen formed less than 10 seconds after addition of the stabilizing reagent and was stable for more than 20 minutes.
  • a blood sample is stained with an aqueous dye solution at a 10:1 (blood:dye) ratio for a few minutes. 28 ⁇ L of the stained blood sample is aspirated to a hematology analyzer with the same reagent volumes and reaction times to the regular 5-part differential analysis described in Example III. The sample mixture is analyzed by fluorescence and DC. The major populations, i.e., lymphocytes, monocytes, neutrophils and eosinophils, can be clearly separated.
  • Example II To 14 ⁇ l of a non-peripheral fluid sample, bone marrow, approximately 1200 ⁇ l of the lytic reagent composition of Example I will be added and the mixture will be gently mixed for about 2 to 7 seconds at room temperature (approximately 21° C.). The lysing reaction will be retarded by the addition of about 490 ⁇ l of the stabilizing reagent composition of Example II. The sample mixture will be gently mixed for a few seconds and ready for differential analysis 7 to 18 seconds after the addition of the stabilizing reagent composition. The differential analysis will be conducted on a hematology analyzer described in Example III. The leukocyte subpopulations will be identified and quantified using the scattergrams illustrated in Example III.
  • T8-FITC fluorochrome labeled antibody
  • Example IIa An aliquot of 34 ⁇ l of the incubated blood sample was pipetted into a test tubing. 521 ⁇ l of the lytic reagent composition of Example Ia was added and the mixture was mixed by swirling for 4 seconds at room temperature (approximately 21° C.). The lysing reaction was retarded by adding 206 ⁇ l of the stabilizing reagent composition of Example IIa. The blood mixture was gently mixed for 5 seconds and analyzed on a COULTER® EPICS® XL flow cytometer using light scatter and fluorescence for CD8 positive lymphocytes.
  • the same incubated blood sample was also treated by a commercial reagent system, ImmunoPrep, using a blood sample preparation instrument, COULTER Q-Prep EPICS® Immunology Workstation (manufactured by Coulter Corporation).
  • the treated sample was analyzed on the same flow cytometer.
  • the ImmunoPrep reagent system and method comprise a 3-step sample preparation: (1) adding Immunoprep Reagent A, an acid lyse, to a blood sample to lyse red cells; (2) adding Immunoprep Reagent B, a quench solution, to quench the lysing reagent; (3) adding ImmunoPrep Reagent C, a fixative reagent, to fix white cells.
  • FIG. 17 shows the correlation between the commercial method and the method of the present invention.
  • the correlation coefficient (0.992), as well as the slope and the intercept of the regression line demonstrate an excellent correlation between the two methods for CD8+%.
  • CD4 positive lymphocytes show a scattergram for differentiation of CD4 positive lymphocytes.
  • the CD4 positive lymphocytes bound to the T4 antibody conjugated latex particles showed a shift on light scatter signals on a RF vs. light scatter scattergram.
  • the CD4 positive lymphocytes formed a distinct cluster and separated from the CD4 negative lymphocyte populations and other leukocytes.
  • the CD4+% results obtained using the method of the present invention are consistent with those obtained using conventional fluorescent flow cytometry.

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PCT/US1998/013553 WO1999005523A1 (en) 1997-07-25 1998-06-29 Method for determination of leukocytes and hemoglobin concentration in blood
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